Specific Aim 1. Mobile Axonal Mitochondria Contribute to the Variability of Synaptic Strength (Sun et al., Cell Reports 2013) One of the most notable characteristics of synaptic physiology in the CNS is the wide pulse-to-pulse variability of synaptic strength in response to identical stimulation. A long-standing question for decades is how this variability arises. This raises a fundamental question: Can those motile mitochondria contribute to the pulse-to-pulse variability of presynaptic strength? We demonstrate that the motility of axonal mitochondria correlates with presynaptic variability. Enhancing axonal mitochondrial motility increases the pulse-to-pulse variability, while immobilizing mitochondria reduces the variability. Using dual-channel imaging at single-bouton levels, we further showed that mitochondrial movement, either into or out of presynaptic boutons, significantly influences SV release due to fluctuation of synaptic ATP levels. A motile mitochondrion passing through synapses changes synaptic energy levels and influence synaptic activities. Therefore, the fluctuation of presynaptic ATP levels contributes to the variability of presynaptic strength. Our study revealed, for the first time, that axonal mitochondrial transport is one of the primary mechanisms underlying the presynaptic variation. Specific Aim 2. Parkin-Independent Mechanism Removing Damaged Mitochondria from Axonal Terminals (Cai et al., Current Biology 2012; Lin et al., submitted) Mitochondrial dysfunction and impaired transport emerge as central problems associate with major neurodegenerative disorders. Mature neurons often show delayed Parkin-mediated mitophagy in response to acute mitochondrial depolarization, raising a question as to whether mitophagy is an early mechanism of neuronal mitochondrial quality control. Acute damage to mitochondria unlikely occurs in vivo. We induce mitochondrial stress with physiologically relevant conditions in healthy neurons and study ALS- and AD-linked neurons with chronic mitochondrial defects. We demonstrate that mitochondrial transport is critical for maintaining their integrity under these physiological and pathological conditions. Releasing SNPH from stressed mitochondria selectively enhances their retrograde transport in a manner independent of Parkin, Drp1 and autophagy. Importantly, this protective mechanism is robustly activated in the early pathological stages in ALS-linked hSOD1G93A spinal motor neurons and AD-linked hAPP cortical neurons. Chronic mitochondrial damage leads to the depletion of SNPH over disease progression, thus compromising this stress response in adult ALS and AD mouse models and patient brains. Our study provides a new mechanistic insight into the maintenance of axonal mitochondrial quality under physiological stress and early disease conditions through coordination with a mitochondrial anchoring protein. Specific Aim 3. Enhancing Mitochondrial Transport Facilitates Axon Regeneration by Rescuing Energy Deficits (Zhou et al., JCB 2016) While young neurons possess robust axon growth during early development, mature CNS axons typically fail to regrow after injury. While regeneration is a highly energy-demanding process, axonal mitochondrial transport progressively declines with maturation, thus raising a fundamental question as to whether mitochondrial transport is necessary to meet enhanced metabolic requirements during regeneration. By applying microfluidic culture system, which allows physical separation of axons from cell bodies and dendrites, we reveal that reduced mitochondrial motility and energy deficits in injured axons are intrinsic mechanisms controlling regrowth in mature neurons. Axotomy induces acute mitochondrial damage and ATP depletion in injured axons. Thus, mature neuron-associated increase in SNPH and decrease in mitochondrial transport cause local energy deficits. Strikingly, enhancing mitochondrial transport facilitates regenerative capacity by replenishing healthy mitochondria in injured axons, thereby rescuing energy deficits. An in vivo sciatic nerve crush study further shows that enhanced mitochondrial transport in snph KO mice accelerates axon regeneration. Therefore, our study suggests a new target for stimulating axon regeneration and functional recovery after nerve injury and disease. Specific Aim 4. Endo-lysosomal Deficits Augment Mitochondria Pathology in Spinal Motor Neurons of Asymptomatic fALS Mice (Xie et al., Neuron 2015) One pathological hallmark in ALS motor neurons (MNs) is axonal accumulation of damaged mitochondria. Proper clearance of those mitochondria via mitophagy may serve as an early protective mechanism. A fundamental question remains: does reduced degradation of those mitochondria due to impaired autophagy-lysosomal system contribute to mitochondrial pathology? We reveal MN-targeted progressive lysosome defects in the hSOD1G93A mice starting as early as postnatal day 40 (P40), accompanied by aberrant accumulation of damaged mitochondria engulfed by autophagosomes in axons. Our in vitro and in vivo studies demonstrate that endo-lysosomal transport is crucial to maintain mitochondrial integrity and MN survival. Such deficits are attributable to impaired retrograde transport of late endosomes by mutant hSOD1G93A, which interferes with dynein-snapin (motor-adaptor) coupling, thus reducing the recruitment of dynein motors to the organelles for transport. These deficits can be rescued by elevated snapin expression. AAV9-snapin injection in hSOD1G93A mice reverses mitochondria pathology, reduces MN loss, and ameliorates the fALS-linked disease phenotype. Our study reveals a new cellular target for early therapeutic intervention. Elucidation of this early pathological mechanism is broadly relevant, because defective transport, lysosomal deficits, and mitochondrial pathology are associated with major neurodegenerative diseases including ALS, Huntingtons, Parkinsons and Alzheimers diseases. Specific Aim 5. Organismal Aging Affects Neuronal Mitochondrial Maintenance (Morsci et al., Journal of Neuroscience 2016). Aging is associated with cognitive decline and increasing risk of neurodegeneration. Perturbation of mitochondrial function, dynamics, and trafficking are implicated in the pathogenesis of several age-associated neurodegenerative diseases. Despite this fundamental importance, the critical understanding of how organismal aging affects lifetime neuronal mitochondrial maintenance remains unknown-particularly in physiologically relevant context. To address this issue, we performed a comprehensive in vivo analysis of age-associated changes in mitochondrial morphology, density, trafficking, and stress resistance in individual Caenorhabditis elegans neurons throughout adult life. Adult neurons display three distinct stages of increase, maintenance and decrease in mitochondrial size and density during adulthood. Mitochondrial trafficking in the distal neuronal processes declines progressively with age starting from early adulthood. In contrast, genetic long-lived daf-2 mutants exhibit delayed age-associated changes in mitochondrial morphology, constant mitochondrial density, and maintained trafficking rates during adulthood. Reduced mitochondrial load at late adulthood correlates with decreased mitochondrial resistance to oxidative stress. Revealing aging-associated changes in neuronal mitochondrial maintenance in vivo is an essential precedent that will allow future elucidation of the mechanistic causes of mitochondrial aging profile. Thus, our study establishes the critical foundation for the future analysis of cellular pathways and genetic and pharmacological factors regulating mitochondrial maintenance in aging- and disease-relevant conditions.